Design of a calcium-binding protein with desired structure in a cell adhesion molecule.

Ca 2+ , a signal of life and death, controls numerous cellular processes through interactions with proteins. An effective approach to understanding the role of Ca 2+ is the design of a Ca 2+ -binding protein with predicted structural and functional properties. To design de novo Ca 2+ -binding sites in proteins is challenging due to the high coordination numbers and the incorporation of charged ligand residues, in addition to Ca 2+ -induced conformational change. Here, we demonstrate the successful design of a Ca 2+ -binding site in the non-Ca 2+ -binding cell adhesion protein CD2. This designed protein, Ca.CD2, exhibits selectivity for Ca 2+ versus other di- and monovalent cations. In addition, La 3+ (K d 5.0 μM) and Tb 3+ (K d 6.6 μM) bind to the designed protein somewhat more tightly than does Ca 2+ (K d 1.4 mM). More interestingly, Ca.CD2 retains the native ability to associate with the natural target molecule. The solution structure reveals that Ca.CD2 binds Ca 2+ at the intended site with the designed arrangement, which validates our general strategy for designing de novo Ca 2+ -binding proteins. The structural information also provides a close view of structural determinants that are necessary for a functional protein to accommodate the metal-binding site. This first success in designing Ca 2+ -binding proteins with desired structural and functional properties opens a new avenue in unveiling key determinants to Ca 2+ binding, the mechanism of Ca 2+ signaling, and Ca 2+ -dependent cell adhesion, while avoiding the complexities of the global conformational changes and cooperativity in natural Ca 2+ -binding proteins. It also represents a major achievement toward designing functional proteins controlled by Ca 2+ binding.